Crystal structure of activated CheY - Comparison with other activated receiver domains

The crystal structure of BeF3--activated CheY, with manganese in the magnes ium binding site, was determined at 2.4-Angstrom resolution. BeF3- bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and s alt bridge with Thr(87) and Lys(109), respectively. The six coordination si tes for manganese are satisfied by a fluorine of BeF3-, the side chain oxyg ens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water m olecules. All of the active site interactions seen for BeF3-- CheY are also observed in P-Spo0A(r), Thus, BeF3- activates CheY as well as other receiv er domains by mimicking both the tetrahedral geometry and electrostatic pot ential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta -strand beta4 and helix H4, The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). Thi s hydrogen bond appears to stabilize the active conformation of the beta4/H 4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, exce pt in the loops, with the largest changes occurring in the beta4/H4 loop. T his region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM, T he pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF3--CheY, P -Spo0A(r) P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) N zeta and the phosphate or its equivalent, beryllofluoride, This suggests t hat, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Ty r coupling), formation of the Lys(109)-PO3- salt bridge is directly involve d in the activation of receiver domains generally.